The transport of hidtidine in Salmonella typhiphimurium and Escherichia coli requires 4 proteins: a histidine-binding, periplasmic protein, J, and 3 membrane-bound components, Q, M, and P. The operon which codes for these proteins has been fully sequenced. The P protein has been shown to contain a nucleotide-binding site which is postulated to be involved in energy-coupling in transport. The architectural organization of J, Q, M, and P will be analyzed by the use of cross-linking and affinity labelling reagents, proteolytic digestion from either side of the membrane, radioactive labelling with membrane-impermeant reagents. Membrane vesicles will be reconstituted for transport by addition of J and used for studying the energy-coupling and the membrane organization of the permease. The nucleotide-binding site of the P protein will be characterized and an enzymatic function (such as ATP hydrolysis) will be sought. The membrane-bound components will be purified, characterized biochemically and used for reconstitution experiments in vesicles and liposomes. For this purpose, antibodies to each of the proteins will be prepared, either by using Beta-galactosidase-fusion proteins or by a variety of other approaches. The mechanism of energy coupling in whole cells and vesicles will be explored since there is considerable confusion concerning the nature of energy coupling (pmf or substrate-level phosphorylation) for periplasmic systems. It is expected that by using a thoroughly characterized periplasmic system (such as the histidine permease) and well characterized mutants in unc (ATPase) and in genes responsible for producing acetylphosphate (ack and pta) it should be possible to clear up this conflict. A strong genetic approach will be pursued to help understand energy coupling. Since all periplasmic systems seem to be similarly organized the relationship between unrelated systems will be explored by computer analysis and by genetic techniques; replacement of a defective transport component with a component from an unrelated system will be selected genetically. The regulation of the transport operon and of the neighboring argT gene by nitrogen availability will be explored. The DNA-binding sites for the regulatory ntrC protein will be established by filter-binding assay and footprinting. Our hypothesis that the ntrC protein might act as an antiterminator will be tested by determining the termination site of transcripts in the presence and absence of the ntrC protein, both in vivo and in vitro. Nitrogen regulatory mutants will be created both in vivo and by in vitro recombinant DNA mutagenesis.